Fusion of a chitin-binding domain to an antibacterial peptide to enhance resistance to Fusarium solani in tobacco (Nicotiana tabacum).

Abstract

An antibacterial peptide-encoding gene from alfalfa seeds, alfAFP, was fused to the C-terminal part of chitin-binding domain (CBD) of the rice chitinase-encoding gene (CBD-alfAFP) and introduced to tobacco by Agrobacterium-mediated transformation. Polymerase chain reaction (PCR) technique was used to confirm the integration of the recombinant CBD-alfAFP encoding gene in transgenic tobacco plants. A number of transgenic lines and a non-transgenic control plant were selected for further molecular analyses. The result of analyzing the transgenic plants by semi-quantitative RT-PCR showed that the recombinant gene is expressed in transgenic plants and there is a difference between the transgenic plants in terms of the level of CBD-alfAFP expression. The total protein was extracted from a few selected transgenic plants and used to evaluate the antibacterial/antifungal of recombinant protein activity against some important plant and human pathogens. The results of this experiment showed that the total protein extract obtained from transgenic lines significantly (P < 0.05) inhibited the growth of various bacteria and fungi compared to the non-transgenic plants. Transgenic lines showed a significant (P < 0.01) difference considering their ability to inhibit bacterial and fungal pathogens growth. The recombinant CBD-alfAFP protein significantly (P < 0.01) increased the resistance of the transgenic plants against Fusarium solani. Transgenic lines showed no significant wilting symptoms and obvious wilting symptoms were not observed even 30 days post-inoculation (dpi) with Fusarium solani spores. These results suggest that transgenic tobacco plants are resistant to Fusarium solani wilt and fusion of CBD to the alfAFP antimicrobial peptide is an efficient approach to control fungal diseases.